Introduction
Scaffold-based vascular tissue engineering aims to regenerate vascular tissue for the replacement of diseased small-calibre blood vessels (diameter less than 6mm). Collagen gel is a commonly used scaffold due to its biological properties including a high potential for supporting and guiding vascular cells in the regeneration process. The approach we privileged consists in first reproducing the media, which provide the high elasticity properties of the vessel wall, thus making it an essential and effective component for blood and nutrients transportation. Starting from an original method, previously reported, for processing collagen and smooth muscle cells (SMCs), the overall goal of this project was to design and develop an endothelialised two layers collagen cell-based scaffold in a disc shape. The underlying layer is composed of fibroblasts (FBs) seeded within collagen. The upper layer is composed of SMCs seeded within collagen and ECs are seeded on this construct. This construct is finally expected to provide vascular tissue remodeling due to cells/cells and cells/matrix interactions and to produce an engineered tissue with properties close to those of blood vessel walls. It is also expected to provide a valid in vitro model for further studies of vascular patho-physiology.

Materials and Methods

Cells culture: ECs were isolated by trypsin treatment of human umbilical cords vein (HUVEC) and expanded in HyClone Media M199/EBSS (Fisher). SMCs were isolated from human umbilical cord artery (HUASMC). Initially, the Wharton's jelly that surrounds the arteries was carefully removed by cutting with scissors. The endothelial layer was removed by scraping the artery intima and afterwards, the arteries were cut to rectangle pieces using scissors and finally placed in a Petri dish with M199 medium. After two weeks, the pieces of the artery were removed and the cells were expanded. Aortic adventitial fibroblasts were purchase from LONZA (Walkersville, MD USA).
Collagen-based gels preparation: Type I collagen was extracted from rat-tail tendons and solubilized in acetic acid solution (0.02 N) at a concentration of 4 g/L according to a protocol previously describe. The collagen solution (2 g/L) was mixed with DMEM and SMCs or FBs (10^6cells/ml), NaOH (15 mM), and Hepes (20 mM) in deionized water. This mixture was then poured in a specific mold and then let jellify for 30 min at room temperature. Then ECs were seeded on the surface at 10^5cells/cm2 and the construct was incubated at 37°C.
Immunofluorescence staining: After 1, 3 or 7 days of maturation, collagen seeded gels were washed twice with PBS and then fixed with 3.7% formaldehyde for 20 min. Gels samples were then permeabilized with 0.5% Triton-X100 for 3 min at room temperature. Gels samples were then additionally incubated with the first the antibody, VE-Cadherin (Abcam), then with the secondary antibody, Alexa 488 (Invitrogen) and Rhodamine phalloidin (Sigma) and Dapi (Invitrogen). The cellular distribution of the fluorescent was assessed using an Olympus BX51 microscope. Images were captured with a Q imaging RETIGA EXI digital camera driven by Image pro express software.
Histochemistry: In order to evaluate the cell distribution in the gel and eventual cell/matrix alignment, histology was performed. Gels samples were rinsed with PBS and fixed in 3.7% formaldehyde, then embedded in paraffin and 10 µm thick sections were sliced and stained with a modified Masson's trichrome. Briefly, sections were deparaffinized with toluene and rehydrated with graded alcohol then refixe in Bouin solution. Three different dyes were used in order to differentiate between cells and extracellular matrix: (1) Weigert's iron hematoxylin solution was used for nuclei staining (dark); (2) acid fuchsin and Xylidine ponceau solution was used for cells cytoplasms (red); and 3) Light green SF yellowish solution for collagen (green).

Results

After 24hrs of growth, the non-circular morphology of endothelilals cells shown by immunofluorescence staining (fig 1) suggests that the cells are growing and proliferating to form a monolayer on top of the collagen gel layers. The presence of SMCs in the background, inside the gel was confirmed by the 3D confocal image (fig 2). Histological staining on transversal section of collagen gels shows the two layers of collagen, one containing SMCs and the others FBs, nested one within the other. After one week of culture, cells density in the gels increases because of gels compaction due to SMCs activities while the ECs layer is shown to remain intact. The alignement of cells due to the specific mold anchoring retaining the compaction in one direction was also observed.

Discussion and Conclusion

Tri-culture of vascular cells were achieved on collagen scaffold without losing endothelial cells. Furthermore, the cells morphologies shown by immunofluorescence staining suggests that cells are making focal attachments. This ongoing experiment shows that it is possible to do vascular cells tri-culture using collagen gel scaffold. The interaction between cells will enhance the matrix remodeling and the properties of the arterial construct. This presentation will show the seeding parameters of all the cells on or in collagen gel. Characterization of cells localization in the collagen matrix will be also shown. Finaly, hemocompatible test will be performed on this construct.
Figure 1: Immunofluorescence staining of cells organization right after 24h of static culture. HUVECs are stained for VE-Cadherin in green, f-actin of HUASMCs with Rhodamin Phalloidin in red and nuclei with Dapi in blue.Figure 2: 3D confocal image of the construct after 24h of static culture. HUVECs are stained for VE-Cadherin in green, f-actin of HUASMCs with Rhodamin Phalloidin in red and nuclei with Dapi in blue.

Acknowledgements

CL was awarded of a PhD Doctoral Scholarship from NSERC CREATE Program in Regenerative Medicine (http://www.ncprm.ulaval.ca).